Endothelium-independent vasodilation effect of di- and tri-peptides in thoracic aorta of Sprague-Dawley rats

The goal of this study was to elucidate the structure-activity relationship for vasodilating peptides and their underlying mechanism. In this study, we synthesized 62 di- and tri-peptides having aromatic amino acid residues (Tyr, Trp and Phe). Among them, only 4 peptides (HW, WH, WL and WV) evoked an apparent vasodilating effect in 50 mM KCl-contracted aortic rings in the descending order of WH>HW>WL>WV; WH showed the vasodilating activity with an EC50 of 3.4 mM. Within our experimental results, it seems likely that Trp residue at the N-terminal would play a role in eliciting vasodilating effect. No appearance of vasodilating effect for stereoisomers of WH with D-configuration revealed that the vessel would recognize the L-configuration of WH. The presence of angiotensin I-converting enzyme (ACE) inhibitor (50 nM enalaprilat) did not affect the WH-induced vasodilating effect, though WH showed a slight ACE inhibitory activity (IC50: 93 microM). The effect was also observed in the endothelium-denuded aortic rings. In contrast, WH provoked a significant displacement to the right in the vasodilating curve in the presence of 30 nM verapamil, while no shift was observed in the presence of 2.5 nM nifedipine. These results indicate that WH, a vasodilating di-peptide, would exert a vasodilation via the suppression of Ca2+ influx into KCl-induced depolarized aortic rings. The present study also suggests that the binding site of WH to the Ca2+ channel may be competitive to that of phenylalkylamine-type voltage-gated L-type Ca2+ channel blocker.     

The vasodilating effect of a Hintonia latiflora extract with antidiabetic action

In the present study, it is shown for the first time that an extract of Hintonia latiflora (HLE) which is used as an antidiabetic herbal medicine, is not only able to decrease blood glucose concentration but additionally exerts a vasodilating effect. Accordingly, this extract might have a positive influence on diabetes-associated dysfunction of blood vessels.The vasodilating effect was demonstrated in vitro in aortic rings of guinea pigs as well as in vivo in rabbits. Aortic rings pre-contracted with noradrenaline (NA) could completely be relaxed by HLE (EC₅₀: 51.98 mg/l). In contrast, potassium-induced contractions were not diminished by HLE. Therefore, it can be suggested that the vasodilating effect of HLE is primarily the result of an inhibition of G protein-induced increase in intracellular calcium and not of a blockade of voltage-operated L-type calcium channels.The neoflavonoid coutareagenin (COU), a constituent of HLE which in part is responsible for the blood glucose-lowering effect of HLE, also relaxed NA-induced contractions of aortic rings (EC₅₀: 32.55 mg/l) and only weakly inhibited potassium-induced contractions.Experiments in aortic rat cells revealed that calcium transients evoked by vasopressin were suppressed by 60 mg/l COU supporting the idea of an inhibition of G protein-induced intracellular calcium release by a constituent of HLE.To study the effect of HLE on vascular tone under in vivo conditions, ultrasound measurements were carried out in conscious rabbits which received a single oral dose of HLE. Under the influence of HLE, a vasodilation combined with a lowering of blood flow velocity could be observed in the abdominal aorta and the common carotid artery. Additionally, a decrease in blood glucose concentration in the HLE group occurred.The combination of a blood glucose-lowering with a vasodilating effect may be helpful for reducing angiopathies, typical long-term complications in patients with diabetes mellitus.     

双环醇减轻超氧阴离子诱导的大鼠胸主动脉舒张功能损伤

目的：探讨双环醇（bicyclol）对超氧阴离子（O2）诱导的血管舒张功能损伤的影响。方法：采用离体器官灌流技术,观察bicyclol对离体大鼠胸主动脉环张力的影响。采用焦酚（O2的供体）建立O2损伤模型,观察bicyclol预孵育对氧化应激损伤后血管内皮依赖性舒张功能的改善作用。结果：bicyclol（10-8~10-5mol/L）对由苯肾上腺素预收缩的内皮完整主动脉环产生舒张作用,该作用可被NO合酶抑制剂L-NAME和环氧化酶抑制剂吲哚美辛阻断。500μmol/L焦酚可引起乙酰胆碱诱导的主动脉环内皮依赖性舒张反应减弱,bicyclol（10-5mol/L）预孵育45 min可减轻焦酚的损伤作用。对于吲哚美辛处理的主动脉环,bicyclol（10-5mol/L）可抑制焦酚所致的血管舒张反应降低,但这一效应未见于L-NAME处理的主动脉环。结论：bicyclol具有内皮依赖性舒血管作用,并能对抗O2引起的血管舒张功能损伤,该作用通过NO途径介导。     

Activation of apoalkaline phosphatase by serum albumin with Zn2+ in rat hepatoma cells.

Reuber rat hepatoma cells (R-Y121B) cultured at 0.5% serum accumulated apoalkaline phosphatase in intact cells. When R-Y121B cells were cultured in the presence of bovine serum albumin, alkaline phosphatase activity increased in the cells, and the associated increase in enzyme activity differed amongst bovine serum albumin preparations. The treatment of bovine serum albumin with activated charcoal not only enhanced the effect of serum albumin on alkaline phosphatase activity, but also cancelled the differences due to different preparations of serum albumin. In contrast, no effect from serum albumin was observed in the increase of alkaline phosphatase activity in R-Y121B cell homogenates incubated at 37 degrees C. The activated-charcoal treatment of bovine serum albumin increased the amount of Zn2+ bound to the protein. When R-Y121B cells were cultured with bovine serum albumin, the concentration of Zn2+ in the cytosol fraction slightly increased. However, the effect of serum albumin on Zn2+ concentration in the cytosol fractions was independent of charcoal treatment. It was concluded that serum albumin with Zn2+ induces the activation of apoalkaline phosphatase due to Zn2+ binding.     

Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: the response to glycated and nonglycated BSA is lost in metabolic syndrome

The effects of nonglycated bovine serum albumin (BSA) and advanced glycosylation end products of BSA (AGE-BSA) on vascular responses of control and metabolic syndrome (MS) rats characterized by hypertriglyceridemia, hypertension, hyperinsulinemia, and insulin resistance were studied. Albumin and in vitro prepared AGE-BSA have vascular effects; however, recent studies indicate that some effects of in vitro prepared AGEs are due to the conditions in which they were generated. We produced AGEs by incubating glucose with BSA for 60 days under sterile conditions in darkness and at 37 degrees C. To develop MS rats, male Wistar animals were given 30% sucrose in drinking water since weanling. Six month old animals were used. Blood pressure, insulin, triglycerides, and serum albumin were increased in MS rats. Contraction of aortic rings elicited with norepinephrine was stronger. There were no effects of nonglycated BSA or AGE-BSA on contractions in control or MS rats; however, both groups responded to L-NAME, an inhibitor of nitric oxide synthesis. Arterial relaxation induced using acetylcholine was smaller in MS rats. Nonglycated BSA and AGE-BSA significantly diminished relaxation in a 35% in the control group but the decrease was similar when using nonglycated BSA and AGE-BSA. This decrease was not present in the MS rats and was not due to increased RAGEs or altered biochemical characteristics of BSA. In conclusion, both BSA and AGE-BSA inhibit vascular relaxation in control artic rings. In MS rats the effect is lost possibly due to alterations in endothelial cells that are a consequence of the illness.     

Complexes of Cu2+ with pyridoxal-5'-phosphate and human and bovine serum albumin

The copper ion Cu2+ bound to serum albumin in the most strong center stabilizes aldimine bonds formed by PLP with epsilon-NH2 group of 4-Lys and alpha-NH2 1-Asp. The stoichometric ratio of the ternary albumin-PLP-Cu2+ complex is 1:2:1. The imidazole rings of histidine residues are involved in binding of copper ions in the first, second, third centers of the albumin molecule. In this case copper ions increase the binding of PLP with the protein stabilizing Schiff bases produced by epsilon-NH2 group of lysine and PLP. The cooper ion bound to serum albumin in the most strong center forms two types of complexes: with rhombic environment in neutral and alkaline media and axial one at pH less than 5,0. On formation of the ternary complex with PLP the rhombic environment is changed to axial.     

Effect of Bovine Serum Albumin Incubation on Lettuce Chloroplasts Digested by Trypsin

1. Bovine serum albumin stimulates the DCIP photoreduction activity of lettuce chloroplasts which has been treated with trypsin. When these chloroplast preparations were washed by tricine buffer such "reversible action" can still be obtained. It is possible that bovine serum albumin may be incorporated into trypsin destroyed site of the membrane. 2. Trypsin-induced CCCP inhibitory effect on DCIP photoreduction activity is reversed by bovine serum albumin. 3. Bovine serum albumin partially reverses the trypsin-induced unstacking of lettuce chloroplast membranes. 4. After trypsin digestion, there are absorbance decreases around 500–640 nm. Bovine serum albumin has no effect on these absorbance decreases. It is concluded that the membrane-bound proteins responsible for different functions of chloroplast are heterogeneous. The results also show that there are gate and channel near the position of PSⅡ on chloroplast membrane.     

Chemical composition and vasodilatation induced by Cuphea carthagenensis preparations

The aerial parts of Cuphea carthagenensis (Jacq.) J.F. Macbride (Lythraceae) are traditionally employed in Brazil to treat cardiovascular diseases. The aim of this study was to compare preparations of C. carthagenensis aerial parts (aqueous and ethanol extracts, together with derived fractions) with regard to their total phenolic contents and in vitro vasodilating activity. The main flavonoids found in the extracts were isolated and identified as quercetin derivatives. The extracts and fractions showed similar HPLC profiles with the presence of quercetin-5-O-β-glucopyranoside, quercetin-3-O-α-arabinofuranoside and quercetin-3-sulfate in all of them, but marked differences in the contents of flavonoids, proanthocyanidins, tannis and total phenolics. Excepting the aqueous extract, all assayed preparations elicited vasodilatation on pre-contracted rat aortic rings in the range of pIC(50) 4.53±0.03 to 4.98±0.06. Polynomial regression analysis demonstrated the relationship between vasodilating activity and the contents of flavonoids (r(2)=0.5190), proanthocyanidins (r(2)=0.8016), tannins (r(2)=0.8041) and total phenolics (r(2)=0.6226), suggesting the participation of these compounds in the pharmacological effect and their potential use as chemical markers for the species.     

Proton magnetic resonance studies of the binding of an anionic surfactant with a benzene ring to a protein polypeptide with special reference to SDS-polyacrylamide gel electrophoresis.

The binding of an anionic surfactant to a protein polypeptide has been studied by the proton magnetic resonance (PMR) technique to form a part of our studies on the principles of SDS-polyacrylamide gel electrophoresis. Sodium 4-(p-butylphenyl) butane-1-sulfonate (CH3-(CH2)3-0-(CH2)4-SO3-Na+) was employed as an anionic surfactant, and reduced and carbosyamidomethylated (RCAM) bovine serum albumin as a typical protein polypeptide. The binding isotherm of the surfactant to RCAM bovine serum albumin was similar to that of sodium dodecyl sulfate (SDS). The surfactant could replace SDS in SDS-polyacrylamide gel electrophoresis without affecting the wellknown mode of spearation of protein bands. These results gave a sound basis for the assumption that the investigation of the complex between a surfactant with a benzene ring and RCAM bovine serum albumin would provide useful knowledge concerning the principles of SDS-polyacrylamide gel electrophoresis. Aggregation of the aromatic surfactant necessarily brings benzene rings together. A benzene ring is a strong source of the ring current effect on chemical shifts in nuclear magnetic resonance (NMR). Chemical shifts of the surfactant in NMR are, therefore, sensitive to whether the surfactant molecules are single-molecularly dissolved or aggregated. Full advantage was taken of the above fact in the present PMR study of the binding of the surfactant to RCAM bovine serum albumin. The chemical shifts of the phenyl and methyl protons both for the single-molecular and micellar aggregated states were estimated from measurements of the shifts as a function of the surfactant concentration. They shifted to a higher magnetic field on micelle formation, due to the increase of the ring current effect. Corresponding measurements for the complex between the surfactant and RCAM bovine serum albumin gave estimates of the chemical shifts of the phenyl and methyl groups of the surfactant bound to the protein polypeptide. They were found to shift to a magnetic field somewhat higher than that for the micellar state throughout the concentration range of the surfactant examined. These results strongly suggest that the surfactant molecules bind to the protein polypeptide in the form of micelle-like clusters, and that PMR of the groups are further influenced by the diagmagnetic effect of the protein polypeptide present as a core. No appreciable change in the mode of binding, corresponding to the steep increase in the amount of binding in the binding isotherm, was observed from the PMR studies. Taking the observed similarity between SDS and the aromatic surfactant in the binding and the gel electrophoresis into consideration, the present results strongly suggest that SDS also binds to protein polypeptides in the form of micelle-like clusters under the conditions of SDS-polyacrylamide gel electrophoreses, and support our "necklace model".     

Removal by bovine serum albumin of fatty acids from membrane vesicles and its effect on proline transport activity in Escherichia coli.

Bovine serum albumin appreciably stimulated the initial rate and the steady-state level of proline uptake in membrane vesicles, while it had no effect on the oxidase activity for ascorbate-phenazine methosulfate, on which the transport activity depends. Bovine serum albumin showed the strongest stimulatory effect on the transport system among the proteins tested. Three other proteins did not show any effect, while beta-lactoglobulin showed a weaker but appreciable effect on the transport activity. The incubation of membrane vesicles with bovine serum albumin resulted in extensive removal of fatty acids, while none of the other membrane components, including proteins and phospholipids, was removed by this treatment. Fatty acids inhibited the proline transport activity, while the inhibited activity was appreciably restored by incubation with the albumin. An experiment with radioactive fatty acids showed that exogenously-added fatty acids bound firmly to the membrane vesicles and were removed by subsequent incubation with the albumin. The incubation of membrane vesicles for several hours resulted in an increase of fatty acids with a concomitant loss of the transport activity. Subsequent incubation with the albumin resulted in the removal of fatty acids and the partial restoration of the transport activity. Based on these results, it is concluded that bovine serum albumin specifically removed fatty acids from membrane vesicles, resulting in activation of the proline transport system.     

Nitric oxide-mediated vasorelaxation by Rhizoma Ligustici wallichii in isolated rat thoracic aorta.

The vasorelaxant effect of Rhizoma Ligustici wallichii and its possible mechanism of action on the vasomotor tone of the rat thoracic aortic rings were examined in an organ bath. Chloroform extracts of Rhizoma Ligustici wallichii (Ch1LW) elicited a dose-dependent, transient, relaxing response in endothelium-intact rat aorta contracted with norepinephrine (NE). This relaxant effect was abolished by removal of the endothelium and also by pretreatment with nitric oxide synthase inhibitors. Neither a muscarinic receptor antagonist nor a cyclooxygenase inhibitor altered the Ch1LW-induced relaxation. Tetramethylpyrazine, derived from Rhizoma Ligustici wallichii as a potent vasodilating component, induced a complete relaxation in both endothelium-intact and denuded rat aortas contracted by NE, but nitric oxide synthase inhibitors did not affect the relaxation. Ch1LW-induced endothelium-dependent relaxation was mediated by nitric oxide released from the endothelium, and could be caused by component(s) other than tetramethylpyrazine.     

Comparative evaluation of the action of serum proteins on tissue regeneration

Processes of skin and long tubular bones' reparative regeneration under the action of serum albumin and alpha-fetoprotein on the body were studied with the use of EPR and histological methods. Relations of spin probe rotatory mobility in cell suspensions and morphological change of examined tissues are shown. It is established that the biological activity of alpha-fetoprotein is more pronounced than that of serum albumin in the effect on the young connective tissue. The stimulating effect of the embryonic protein is demonstrated only in the first week of reparative process while that of serum albumin appeared much more late.     

Purification and characterization of the serum ferroxidase inhibitor

The inhibitor of the serum ferroxidases, recently detected in rabbit serum, has been purified to homogeneity from human serum by a combination of gel-filtration and ion-exchange chromatography. The molecular weight, chromatographic behavior, electrophoretic mobility, electrofocusing pH, carbohydrate content, and reactivity with anti-human albumin during immunodiffusion indicate that the ferroxidase inhibitor is serum albumin. Copper-binding studies, proteolytic fragmentation studies, and a comparison of the inhibitory potencies of several albumin species which differ in their affinity for copper strongly indicate that albumin elicits its inhibitory effect on the serum ferroxidases by interacting with the functional copper of these enzymes. Kinetic analyses further suggest that albumin competes with substrate (ferrous iron) for binding to the functional copper of the serum ferroxidases.     

Dynamics of initial L-cell adhesion. II. Competition of albumin and serum activating factor

The initial attachment process of L cells is studied by the combined action of serum and albumin. Both the substances are added jointly in the Eagle medium or one of them was adsorbed on the substrate previously. The results show that there are two factors in the serum: one depressing the cell attachment, like albumin, and the other being just opposite. The simple kinetical competition model is suggested to describe the experimental dependence of final level of attachment on the concentrations of serum and albumin. The examination of the thermal resistance of the serum factor is made; the previous heating to 60-100 degrees C increases the depressing effect of serum and albumin.     

Phosphoinositide turnover in human platelets is stimulated by albumin.

The effect of human serum albumin (HSA) on the hydrolysis of phosphatidylinositides in human platelets labeled with myo(3H)inositol was studied. Incubation of platelets with HSA (4 gm/dl) for 10 seconds increased IP2, and IP3, by 169% and 217% respectively. 93% of IP3 accumulated within the first 10 seconds. This effect was also shared by bovine serum albumin, although no changes in IP3 levels occurred with ovalbumin. All albumin species used induced 45Ca+2 release from platelets irrespective of its effect on IP3 accumulation. These findings indicate that albumin may function in biological systems by inducing intracellular signaling.     

Effect of bilirubin and its photodegradation products on the respiration of mitochondria isolated from rat liver

In 0.05--0.1 mmol.l-1 concentration, bilirubin inhibits ADP-activated respiration of isolated liver mitochondria; it has no effect on respiration in the absence of ADP. Bilirubin-induced inhibition of respiration is not abolished by serum albumin, but bilirubin bound to serum albumin and the photodegradation products of bilirubin have no inhibitory effect.     

Activation of delta5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins.

The Δ⁵-3-ketosteroid isomerase (EC 5.3.3.1) of bovine adrenal microsomes is activated as much as 10- to 20-fold by micromolar concentrations of bovine serum albumin. Comparable activations are observed with the serum albumins of 10 other mammalian species, but are not seen with ovalbumin or conalbumin. Evidence that the activation is attributable to the serum albumins, rather than to a small, firmly-bound ligand, is based on: (1) Failure to remove the stimulatory activity from the albumin by chloroform extraction, dialysis, or gel filtration; (2) Destruction of the activity by heating or by trypsin digestion; (3) Precipitation of the stimulatory activity of bovine serum albumin by specific antibody. Bovine serum albumin induces small decreases in the Michaelis constant for Δ⁵-androstene-3,17-dione, but most of the activational effect reflects an increase in the maximum velocity. Low concentrations of Triton X-100, which are without effect on the isomerase activity, prevent the activation by bovine serum albumin.     

The effect of bovine and human serum albumins on the mechanical properties of human erythrocyte membranes

Crystalline bovine serum albumin increased the mechanical resistance of fresh human erythrocytes to lysis by hydrodynamic shear forces. A saturation effect suggests that the bovine alubmin molecules are adsorbed on to a finite number of “attachment sites” on the erythrocyte surface, possibly by displacing human proteins already occupying these sites. A heterogeneous fraction of human serum albumins does not exhibit the same marked protection effect, nor displace adsorbed bovine albumin molecules from the erythrocyte surface. The precise nature and extent of the interaction between any given concentration of either human or bovine serum albumin and the intact erythrocyte membrane depends upon the chronological age of the cell concerned.     

Study of the interaction of human serum albumin with penicillins by means of spin labels]

Interaction of 8 penicillin preparations with human serum albumin was studied with the spin-labels method and a probe. Correlation between the binding level of penicillins with human serum albumin and their effect on the spectrum of EPR of the spin-label attached to albumin was observed only with the use of a hydrophobic probe (radical III). The covalent attached marks and the hydrophobic probe may be used for rapid orienting estimation of pencillin interaction with albumin.     

Continuous culture of rat C6 glioma in serum-free medium

In this communication we describe serum-free culture conditions for the serial propagation of the C6 glioma cell line. The growth rate, saturation density, and morphology of these cells are equivalent to those of their serum-grown counterparts when cultured in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's medium F-12 supplemented with trace elements, insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and a serum-spreading factor (SSF) partially purified from human plasma. The requirement for SSF in the medium can be satisfied by preincubating the tissue culture dishes with SSF. Tissue culture dishes sequentially pretreated with poly-D-lysine and purified cold insouluble globulin will also substitute for this requirement. The fatty acid-free bovine serum albumin/linoleic acid complex increases the growth rate of these cells but has no appreciable effect on their morphology, saturation density, or ability to grow with repeated subculture. The growth stimulation caused by this complex appears to be dependent on the fatty acid, as the fatty acid-free bovine serum albumin alone has no effect on the growth rate. Linoleic acid is cytotoxic in the absence of bovine serum albumin, and the fatty acid-free bovine serum albumin prevents this toxicity. Other fatty acids including oleic, arachidonic, and palmitic only partially substitute for the growth-promoting effect of linoleic acid.